Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Keratinocyte growth factor (KGF) is a potent and specific mitogen for epithelial cells, including the keratinocytes of the skin. We investigated the mechanisms of action of KGF by searching for genes which are regulated by this growth factor in cultured human keratinocytes. Using the differential display RT-PCR technology we identified the gene encoding adenylosuccinate lyase [EC 4.3.2.2] as a novel KGF-regulated gene. Adenylosuccinate lyase plays an important role in purine de novo synthesis. To gain further insight into the potential role of nucleotide biosynthesis in the mitogenic effect of KGF, we cloned cDNA fragments of the key regulatory enzymes involved in purine and pyrimidine metabolism (adenylosuccinate synthetase [EC 6.3.4.4], phosphoribosyl pyrophosphate synthetase [EC 2.7.6.1], amidophosphoribosyl transferase [EC 2.4.2.14], hypoxanthine guanine phosphoribosyl transferase [EC 2.4.2.8] and the multifunctional protein CAD which includes the enzymatic activities of carbamoyl-phosphate synthetase II [EC 6.3.5.59], aspartate transcarbamylase [EC 2.1.3.2] and dihydroorotase [EC 3.5.2.3]). Expression of all of these enzymes was upregulated after treatment with KGF and also with epidermal growth factor (EGF), indicating that these mitogens stimulate nucleotide production by induction of these enzymes. To determine a possible in vivo correlation between the expression of KGF, EGF and the enzymes mentioned above, we analysed the expression of the enzymes during cutaneous wound repair, where high levels of these mitogens are present. Indeed, we found a strong mRNA expression of all of these enzymes in the EGF- and KGF-responsive keratinocytes of the hyperproliferative epithelium at the wound edge, indicating that their expression might also be regulated by growth factors during wound healing.
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PMID:Growth factor-regulated expression of enzymes involved in nucleotide biosynthesis: a novel mechanism of growth factor action. 1059 72

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8.; HPRT) catalyzes the salvage synthesis of inosine-5'-monophosphate (IMP) and guanosine-5'-monophosphate (GMP) from the purine bases hypoxanthine and guanine, respectively. Complete deficiency of HPRT activity is associated with the Lesch-Nyhan syndrome (LNS), characterized by excessive purine production and severe neurological manifestations. The etiology of the metabolic consequences of HPRT deficiency is clarified, but that of the neurological manifestations is not yet understood. HPRT-deficient mice represent an experimental animal model of LNS. In search for a possible metabolic abnormality in LNS brains, connecting the neurological deficit to HPRT deficiency, the purine and pyrimidine nucleotide content of cultured neurons, prepared from HPRT-deficient transgenic mice, was now determined. The HPRT-deficient neuronal cultures exhibited a significantly elevated content of the pyrimidine nucleotides UTP (1.33-fold the normal level, p = 0.0002) and CTP (1.28-fold the normal level, p = 0.02), but normal content of the purine nucleotides ATP and GTP. This abnormality in neuronal pyrimidine nucleotide content may be associated with the pathophysiology of the neurological deficit in LNS.
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PMID:Elevated UTP and CTP content in cultured neurons from HPRT-deficient transgenic mice. 1085 40

We recently showed that an increased supply of purine nucleotides increased the growth rate of cultured fibroblasts. To understand the mechanism of the growth rate regulation, CHO K1 (a wild type of Chinese hamster ovary fibroblast cell line) and CHO ade (-)A (a cell line deficient in amidophosphoribosyltransferase, a rate-limiting enzyme of the de novo pathway) were cultured under various conditions. Moreover, a defective de novo pathway in CHO ade (-)A cells was exogenously restored by 5-amino-4-imidazole-carboxamide riboside, a precursor of the de novo pathway. The following parameters were determined: the growth rate of CHO fibroblasts, the metabolic rate of the de novo pathway, the enzyme activities of amidophosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, the content of intracellular nucleotides, and the duration of each cell-cycle phase. We concluded the following: (i) Purine de novo synthesis, rather than purine salvage synthesis or pyrimidine synthesis, limits the growth rate. (ii) Purine nucleotides are synthesized preferentially by the salvage pathway as long as hypoxanthine is available for energy conservation. (iii) The GTP content depends on the intracellular ATP content. (iv) Biosynthesis of purine nucleotides increases the growth rate mainly through ATP production and promotion of the G(1)/S transition.
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PMID:The rate of cell growth is regulated by purine biosynthesis via ATP production and G(1) to S phase transition. 1087 58

Triplex forming oligonucleotides (TFOs) are of interest because of their potential for facile gene targeting. However, the failure of TFOs to bind target sequences at physiological pH and Mg(2+) concentration has limited their biological applications. Recently, pyrimidine TFOs with 2'-O-aminoethyl (AE) substitutions were shown to have enhanced kinetics and stability of triplex formation (Cuenoud, B., Casset, F., Husken, D., Natt, F., Wolf, R. M., Altmann, K. H., Martin, P., and Moser H. E. (1998) Angew. Chem. Int. Ed. 37, 1288--1291). We have prepared psoralen-linked TFOs with varying amounts of the AE-modified residues, and have characterized them in biochemical assays in vitro, and in stability and HPRT gene knockout assays in vivo. The AE TFOs showed higher affinity for the target in vitro than a TFO with uniform 2'-OMe substitution, with relatively little loss of affinity when the assay was performed in reduced Mg(2+). Once formed they were also more stable in "physiological" buffer, with the greatest affinity and stability displayed by the TFO with all but one residue in the AE format. However, TFOs with lesser amounts of the AE modification formed the most stable triplexes in vivo, and showed the highest HPRT gene knockout activity. We conclude that the AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.
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PMID:Targeted gene knockout by 2'-O-aminoethyl modified triplex forming oligonucleotides. 1138 47

The relationship between a complete deficiency of the purine enzyme hypoxanthine-guanine phosphoribosyltransferase and the neurobehavioural abnormalities in Lesch-Nyhan disease remains an enigma. In vitro studies using lymphoblasts or fibroblasts have evaluated purine and pyrimidine metabolism with conflicting results. This study focused on pyridine nucleotide metabolism in control and Lesch-Nyhan fibroblasts using radiolabelled salvage precursors to couple the extent of uptake with endocellular nucleotide concentrations. The novel finding, highlighted by specific culture conditions, was a marked NAD depletion in Lesch-Nyhan fibroblasts. ATP and GTP were also 50% of the control, as reported in lymphoblasts. A 6-fold greater incorporation of [(14)C]nicotinic acid into nicotinic acid- adenine dinucleotide by Lesch-Nyhan fibroblasts, with no unmetabolized substrate (20% in controls), supported disturbed pyridine metabolism, NAD depletion being related to utilization by poly(ADP-ribose) polymerase in DNA repair. Although pyrimidine nucleotide concentrations were similar to controls, Lesch-Nyhan cells showed reduced [(14)C]cytidine/uridine salvage into UDP sugars. Incorporation of [(14)C]uridine into CTP by both was minimal, with more than 50% [(14)C]cytidine metabolized to UTP, indicating that fibroblasts, unlike lymphoblasts, lack active CTP synthetase, but possess cytidine deaminase. Restricted culture conditions may be neccesary to mimic the situation in human brain cells at an early developmental stage. Cell type may be equally important. NAD plus ATP depletion in developing brain could restrict DNA repair, leading to neuronal damage/loss by apoptosis, and, with GTP depletion, affect neurotransmitter synthesis and basal ganglia dopaminergic neuronal systems. Thus aberrant pyridine nucleotide metabolism could play a vital role in the pathophysiology of Lesch-Nyhan disease.
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PMID:Severe pyridine nucleotide depletion in fibroblasts from Lesch-Nyhan patients. 1199 69

Triple helix forming oligonucleotides (TFOs) that bind chromosomal targets in living cells may become tools for genome manipulation, including gene knockout, conversion, or recombination. However, triplex formation by DNA third strands, particularly those in the pyrimidine motif, requires nonphysiological pH and Mg(2+) concentration, and this limits their development as gene-targeting reagents. Recent advances in oligonucleotide chemistry promise to solve these problems. For this study TFOs containing 2'-O-methoxy (2'-OMe) and 2'-O-(2-aminoethyl) (2'-AE) ribose substitutions in varying proportion have been constructed. The TFOs were linked to psoralen and designed to target and mutagenize a site in the hamster HPRT gene. T(m) analyses showed that triplexes formed by these TFOs were more stable than the underlying duplex, regardless of 2'-OMe/2'-AE ratio. However, TFOs with 2'-AE residues were more stable in physiological pH than those with only 2'-OMe sugars, as a simple function of 2'-AE content. In contrast, gene knockout assays revealed a threshold requirement--TFOs with three or four 2'-AE residues were at least 10-fold more active than the TFO with two 2'-AE residues. The HPRT knockout frequencies with the most active TFOs were 300-400-fold above the background, whereas there was no activity against the APRT gene, a monitor of nonspecific mutagenesis.
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PMID:Minimum number of 2'-O-(2-aminoethyl) residues required for gene knockout activity by triple helix forming oligonucleotides. 1205 3

The purine nucleoside cycle is a cyclic pathway composed of three cytosolic enzymes, hypoxanthine-guanine phosphoribosyltransferase, IMP-GMP specific 5'-nucleotidase, and purine-nucleoside phosphorylase. It may be considered a 'futile cycle', whose net reaction is the hydrolysis of 5-phosphoribosyl-1-pyrophosphate to inorganic pyrophosphate and ribose 1-phosphate. The availability of a highly purified preparation of cytosolic 5'-nucleotidase prompted us to reconstitute the purine nucleoside cycle. Its kinetics were strikingly similar to those observed when dialyzed extracts of rat brain were used. Thus, when the cycle is started by addition of inorganic phospate (Pi) and hypoxanthine or inosine (the 'inosine cycle'), steady-state levels of the intermediates are observed and the cycle 'turns over' as far as 5-phosphoribosyl-1-pyrophosphate is being consumed. In the presence of ATP, which acts both as an activator of IMP-GMP-specific 5'-nucleotidase and as substrate of nucleoside mono- and di-phosphokinases, no IDP and ITP are formed. The inosine cycle is further favored by the extremely low xanthine oxidase activity. Evidence is presented that ribose 1-phosphate needed to salvage pyrimidine bases in rat brain may arise, at least in part, from the 5-phosphoribosyl-1-pyrophosphate hydrolysis as catalyzed by the inosine cycle, showing that it may function as a link between purine and pyrimidine salvage. When the cycle is started by addition of Pi and guanine (the 'guanosine cycle'), xanthine and xanthosine are formed, in addition to GMP and guanosine, showing that the guanosine cycle 'turns over' in conjunction with the recycling of ribose 1-phosphate for nucleoside interconversion. In the presence of ATP, GDP and GTP are also formed, and the velocity of the cycle is drastically reduced, suggesting that it might metabolically modulate the salvage synthesis of guanyl nucleotides.
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PMID:The purine nucleoside cycle in cell-free extracts of rat brain: evidence for the occurrence of an inosine and a guanosine cycle with distinct metabolic roles. 1278 25

Various enzyme defects in the metabolic pathways of purines and pyrimidines are known, which result in different diseases occurring in children. They mainly affect kidney function, central nervous system, immunological and blood system. For example, complete deficiency of HPRT (hypoxanthine-guanine-phosphoribosyl-transferase) causes the Lesch Nyhan syndrome, which is characterized by hyperuricemia, mental retardation, choreoathetosis and compulsive self-mutilation. XDH deficiency (xanthine-dehydrogenase) causes in arthropathia and myopathia. For screening for these and other enzyme defects, urinary purine and pyrimidine excretion is considered a simple diagnostic tool. The purpose of the present study was to establish a reverse phase HPLC screening method for urinary purines and pyrimidines and to establish age related reference ranges in children for the urinary excretion of orotic acid, uracile, pseudouridine, uric acid, hypoxanthine, xanthine, thymine, 7-methylguanine, inosine, guanosine and adenosine.
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PMID:Purine and pyrimidine metabolites in children's urine. 1292 81

The purine analogue, allopurinol, has been in clinical use for more than 30 years as an inhibitor of xanthine oxidase (XO) in the treatment of hyperuricemia and gout. As consequences of structural similarities to purine compounds, however, allopurinol, its major active product, oxypurinol, and their respective metabolites inhibit other enzymes involved in purine and pyrimidine metabolism. Febuxostat (TEI-6720, TMX-67) is a potent, non-purine inhibitor of XO, currently under clinical evaluation for the treatment of hyperuricemia and gout. In this study, we investigated the effects of febuxostat on several enzymes in purine and pyrimidine metabolism and characterized the mechanism of febuxostat inhibition of XO activity. Febuxostat displayed potent mixed-type inhibition of the activity of purified bovine milk XO, with Ki and Ki' values of 0.6 and 3.1 nM respectively, indicating inhibition of both the oxidized and reduced forms of XO. In contrast, at concentrations up to 100 muM, febuxostat had no significant effects on the activities of the following enzymes of purine and pyrimidine metabolism: guanine deaminase, hypoxanthine-guanine phosphoribosyltransferase, purine nucleoside phosphorylase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. These results demonstrate that febuxostat is a potent non-purine, selective inhibitor of XO, and could be useful for the treatment of hyperuricemia and gout.
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PMID:Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase. 1569 61

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-alpha-d-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP.Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg(2+).PP(i) complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9A resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasma gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T.gondii).
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PMID:The crystal structure of free human hypoxanthine-guanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle. 1599 Jan 11


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